Sterically stabilized carrier for aerosol therapeutics, compositions and methods for treating the respiratory tract of a mammal

ABSTRACT

This invention comprises a sterically stabilized liposome carrier encapsulating a selected drug for the aerosol delivery of the drug effectual in the treatment of the lungs of a mammal, a composition containing the sterically stabilized liposome carrier and the selected drug effective for the treatment of the lungs of a mammal for asthma and inflammation of the lungs of the mammal as an aerosol and a method of, treatment using the composition. The composition provides effective treatment for the longer of a period of time at least twice as long as the selected drug alone or up to at least one week.

RELATED CASES

This application is a continuation-in-part of U.S. Ser. No. 11/287,703filed Nov. 22, 2005 now abandoned entitled “Sterically StabilizedLiposome and Triamcinolone Composition for Treating the RespiratoryTract of a Mammal” by K. S. Konduri, et al which is entitled to andclaims the benefit of the filing date of U.S. provisional applicationNo. 60/632,181 filed Dec. 1, 2004 by K. S. Konduri, et al and of U.S.Ser. No. 10/769,034 filed May 30, 2004 entitled “A Sterically StabilizedCarrier for Aerosol Therapeutic Compositions and Methods for TreatingDiseases of the Respiratory Tract of a Mammal” by K. S. Konduri, et alwhich is entitled to and claims the benefit of the filing dates of U.S.Provisional Nos. 60/498,609 and 60/498,546, both filed Aug. 28, 2003.

FIELD OF THE INVENTION

This invention is directed to a sterically stabilized liposome carriereffective for the aerosol delivery of a drug effectual in the treatmentof a mammal, a composition comprising the sterically stabilized liposomecarrier and a drug effective for the treatment of a mammal which isadministered via the respiratory tract of a mammal as an aerosol and amethod of treatment using the composition. The composition provides,with one dose, effective treatment for the longer of a period of timetwice as long as the effective time for aerosol treatment of the mammalwith a comparable quantity of the drug alone or up to at least sevendays.

BACKGROUND OF THE INVENTION

Asthma is a common disease that causes recurrent symptoms, repeatedhospitalizations and an increased risk of sudden death. It is the mostcommon childhood illness and affects 20 million Americans. According tothe American Academy of Allergy, Asthma & Immunology, asthma causesdirect health care costs in the United States of over $11.5 billionannually. Additionally, in the United States lost productivity addsanother $4.6 billion and drugs prescribed for asthma patients representcosts of over $5 billion annually.

Asthma is characterized by acute bronchial constriction, chronic lunginflammation and airway hyperreactivity which results in chronicinflammation and airway remodeling that leads to progressive andpossibly irreversible airway damage. The most effective therapy focuseson the early stages of the disease before the vicious cycle ofinflammatory changes can become irreparable. The disease usually startsin early childhood and most commonly before five years of age. Thus,appropriate management of asthma in childhood may have a greater impacton the course of the disease than interventions later in life.

The mainstay of asthma treatment therapy is the use of anti-inflammatorydrugs (i.e., inhaled corticosteroids). As a first line therapy forpatients above five years of age, inhaled corticosteroids are usuallygiven via an inhaler twice a day. Patients under five years of age arefrequently given a nebulized form of budesonide (BUD), which is a potentinhaled corticosteroid, given twice a day as a first line therapy.

Although inhaled corticosteroids are very effective in preventing themassive inflammation that occurs with asthma, they do have some majordrawbacks. One is that these drugs must be given at least daily to beeffective. For instance, the effective life of BUD alone in the lungs isno more than one day. This daily dosage requirement may lead tonon-adherence by the patient. Since adherence to daily use of inhaledcorticosteriods by the patient is critical in interrupting the chronicinflammation that occurs in asthma, this becomes a focal issue foreffective therapy. Further the effective use of an inhaler is verytechnique-dependent. Typically only up to about fifteen percent of agiven dose is delivered to the lungs using an inhaler. The inhaledcorticosteroids have a short half-life in the body and have potentialtoxicity when used in higher doses. These are serious disadvantages tothe use of corticosteroid drugs in conventional therapy.

In an abstract published by the present inventors in the Journal ofAllergy Clinical Immunology entitled “Efficacy of Liposome EncapsulatedBudesonide in Experimental Asthma,” February, 2001, Vol. 107, No. 2, itis disclosed that BUD encapsulated in sterically stabilized liposomesprevents asthma inflammation in lower doses given at less frequentdosing intervals by comparison to daily BUD therapy. Test results aresummarized demonstrating an improvement. The abstract does not disclosea suitable sterically stabilized liposome, suitable types of stericallystabilized liposomes or any method for producing a suitable stericallystabilized liposome, for producing BUD encapsulated in a suitablesterically stabilized liposome or a method for administering thesterically stabilized liposome containing BUD.

In view of the likelihood of possible adverse effects with use ofcorticosteroids and the frequency with which the corticosteroids andother drugs are required, a continued effort has been directed to thedevelopment of improved techniques for administering a drug to a mammalvia the respiratory tract of the mammal so that it may be administeredmore effectively and so that the effectiveness of the drug can beachieved using smaller doses and at less frequent dosing intervals.

SUMMARY OF THE INVENTION

The present invention comprises a composition consisting essentially ofa sterically stabilized liposome carrier having a gel-liquid transitiontemperature from about −20° C. to about 44° C. and encapsulating aselected drug effective for aerosol administration to the respiratorytract of a mammal, the sterically stabilized liposome carrier comprisingphosphatidylcholine, phosphatidylglycerol and poly(ethylene glycol), thecomposition being of a particulate size up to about 0.2 to about 5.0microns and the effective to result in deposit of the selected drug inthe airways down to the alveoli and alveolar tight junction uponinhalation of the composition by the mammal and providing an effectivelife for the selected drug in a mammal equal to at least twice theeffective life, or up to a week, of a single dose of the selected drugalone.

The invention further comprises a method for treating a mammal with aselected drug by forming an aerosol of a composition consisting of asterically stabilized liposome carrier encapsulating an effective amountof the selected drug effective for treatment of the mammal. Thesterically stabilized liposome comprising phosphatidylcholine,phosphatidylglycerol, and poly(ethylene glycol), the composition beingof a particulate size up to about 0.2 to about 5.0 microns and effectiveto result in deposit of the selected drug in the airways down to thealveoli and alveolar tight junction having a gel-liquid transitiontemperature from about −20° C. to about 44° C. and providing aneffective life for the drug in the respiratory tract of a mammal equalto at least twice the life of a single dose, or up to a week, of theselected drug alone; and, allowing the mammal to inhale an effectiveamount of the aerosol at selected time intervals.

The invention further comprises a method for treating a mammal with aselected drug by forming an aerosol of a carrier consisting essentiallyof a sterically stabilized liposome carrier encapsulating the selecteddrug effective for treatment of the respiratory tract of a mammal. Thesterically stabilized liposome carrier consists essentially ofphosphatidylcholine and poly (ethylene glycol), the compositionproviding an effective life for the drug in the respiratory tract of amammal equal to at least twice the effective life or up to a week of asingle dose of the selected drug alone; and, allowing the mammal toinhale an effective amount of the aerosol at selected time intervals.

The invention also comprises a method for treating a mammal with aselected drug by forming an aerosol of a composition consisting of asterically stabilized liposome carrier encapsulating an effective amountof a selected drug effective for treatment of the mammal. The stericallystabilized liposome carrier consists essentially of phosphatidylglyceroland poly (ethylene glycol), the composition providing an effective lifefor the drug in the respiratory tract of a mammal equal to at leasttwice the effective life, or up to a week, of a single does of theselected drug alone; and, allowing the mammal to inhale an effectiveamount of the aerosol at selected time intervals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 show graphical and pictorial presentations of a comparison ofthe use of BUD encapsulated in the carrier to the use of BUDencapsulated in the conventional carrier on lung inflammation for themice groups listed in BUD 1;

FIGS. 6-8 show graphical presentations of a comparison of the use of BUDencapsulated in the carrier with free BUD and free carrier givensimultaneously on lung inflammation for the mice groups tested in BUD2;

FIGS. 9-15 show graphical and pictorial presentations of the use of BUDencapsulated in the carrier with and without cholesterol on lunginflammation and AHR for the mice groups tested in BUD 3;

FIGS. 16-18 show graphical presentations of a comparison of the use ofBUD encapsulated in the carrier with and without MPL on lunginflammation and AHR and for the mice groups tested in BUD 4;

FIGS. 19-23 show graphical and pictorial presentations of a comparisonof the use of either BUD or TRI encapsulated in the carrier on lunginflammation and AHR for the mice groups tested in BUD 5;

FIGS. 24-32 show graphical and pictorial presentations of the use ofD-4F encapsulated in the carrier on lung inflammation and AHR for themice groups tested in D-4F;

FIGS. 33-37 show graphical presentations of the use of SLPI encapsulatedin the carrier on lung inflammation and AHR for the mice groups testedin SLIPI.

FIGS. 38-40 show pictures of Electron Microscopy of lung tissues withthe BUD encapsulated in the carrier in comparison with BUD encapsulatedin the conventional carrier in EM.

DESCRIPTION OF PREFERRED EMBODIMENTS

Liposomes are well known materials that comprise primarily phospholipidbilayered vesicles of many types that can encapsulate a variety of drugsand some types are avidly phagocytosed by macrophages in the body. Thevarious interactions of the liposomes can be generalized into fourcategories: (1) exchange of materials, including lipids, lipids andproteins with cell membranes or transfer of encapsulated drugs to thecell; (2) absorption or binding of liposomes to cells; (3) cellinternalization of liposomes by endocytosis or phagocytosis once boundto the cell; and, (4) fusion of bound liposomes with the cell membrane.In all these interactions, there is a strong dependence on lipidcomposition, type of cell, presence of specific receptors and many otherparameters.

Liposomes have been used to provide drugs in mammals, particularly whenit is desired to apply the drugs to specific areas for specificapplications. Liposomes have been used to encapsulate antibiotics,antiviral agents and the like and have been shown to enable enhancedefficacy against a variety of infectious diseases. A major drawback ofconventional liposomes is that they have a relatively short life in amammal body. Most applications have used liposomes in the bloodstream.

To extend the life of liposomes in a mammal body, attempts have beenmade to develop sterically stabilized liposomes, which have a longerlife in a mammal body. Attempts to extend the life of liposomes haveincluded the use of poly(ethylene glycol), natural glycolipids,surfactants, polyvinyl alcohol, polylactic acid, polyglycolic acid,polyvinyl pyrrolidene, polyacrylamine and other materials in variouscombinations with the liposomes in attempts to provide stericallystabilized liposomes, which are effective for drug delivery and whichare compatible with a mammal circulatory system. The most prominentsterically stabilized liposomes utilize distearoylphosphatidylcholine asthe primary phospholipid.

Liposome-encapsulated antibiotics show increased efficacy for treatmentof a variety of infectious diseases. Liposomes have also been consideredfor the delivery of aerosolized asthma medications, such as chromolynsodium and albuterol sulfate. However the potential role of liposomeencapsulation in enhancing the efficacy of inhaled steroid preparationsused in asthma remained unknown at the time of this invention.

Liposomes are characterized by their lipid composition, surface charge,steric interactions and number of lamellae. Conventional liposomes arecomposed of naturally-occurring phospholipids, such asphosphatidylglycerol and phosphatidylcholine mixed with or withoutcholesterol. Although conventional liposomes can encapsulate a varietyof drugs, they are recognized in vivo by the cells of thereticuloendothelial system and are cleared rapidly from the circulation.In addition, incorporation of triamcinolone (TRI) or beclomethasone intoconventional liposomes results in their rapid redistribution and leakagefrom liposomes into the medium.

In contrast to conventional liposomes, sterically stabilized liposomesexhibit increased stability in plasma and decreased uptake by thereticuloendothelial system. Although several studies have reported theuse of conventional liposomes in asthma therapy, sterically stabilizedliposomes have not been investigated as a carrier for the delivery ofanti-inflammatory or other drugs by aerosol administration to therespiratory tract of a mammal. For conciseness the present invention hasbeen discussed and its efficacy shown by treatment of a respiratorytract of a mammal for asthma, although the invention is not limited toasthma treatment or drugs for treatment of the respiratory tract.

For use in the present invention, it has been necessary to producesterically stabilized liposomes which are compatible with a mammalrespiratory system and lungs, adapted for aerosol administration to themammal and which have an extended life in the lungs, respiratory tractand bloodstream. Thus conventional liposomes are not functional for thepurpose of treating the respiratory tract of a mammal for theapplications discussed in this application.

A property of the carriers of the present invention is that the carriersare uniquely adapted for use in the lungs. They have the ability to notdisrupt the composition and function of lung surfactant which provides alateral surface pressure in the lungs which prevents lung collapse. Thusan ideal mixture of lipids in the sterically stabilized liposomes willbe one closest to that of lung surfactant lipids. One such lipidcomposition is DPPC:DPPG:PEG-DSPE (80:15:5). An alternative lipidcomposition is DPPC:DPPG:PEG-DSPE (78:18:4). DPPC is an abbreviation fordipalmitoylphosphatidylcholine. DPPG is an abbreviation fordipalmitoylphosphatidylglycol. PEG is an abbreviation for poly(ethyleneglycol). DPPE is an abbreviation for distearoylphosphatidylethanolamine.The ratios are expressed as molar ratios.

Properties of the carrier of the present invention uniquely adapted toretain the drug for long periods of time are: (1) its composition whichfacilitates the encapsulation of a drug within the bilayer or inside thecarrier; (2) the presence of sufficient amounts of PEGylated (PEG refersto poly(ethylene glycol)) lipids to stabilize and protect the liposomefrom disruption upon exposure to biological milieu, including lungsurfactant and lung surfactant proteins and upon nebulization; and, (3)the presence of an amount of PEGylated lipid sufficient to enable thedrug to remain liposome-associated for a long enough period to beeffective in the lungs.

The sterically stabilized liposomes of the carriers have a compositionsuch that they are readily administered to the mammal as an aerosol andwill remain stable in the presence of serum and in the extra-cellularenvironment. They preferentially localize to the lungs, especially toareas of inflammation as commonly seen in asthma, i.e., in lunginflammation and in the airway hypersensitivity response. A suitable wayto administer the composition of the present invention is via anaerosolization, such as nebulizer. These sterically stabilized liposomesare amenable to nebulization. The combination of these stericallystabilized liposomes with encapsulated drugs useful in the treatment ofmammalian respiratory tract diseases has been shown herein withcorticosteroids: BUD and TRI; monophosphoryl lipid A (MPL); peptides:D-4F (apo1 lipoprotein A-1 mimetic) and Serine Lung Protease Inhibitor(SLPI) for the treatment of lung inflammation and airwayhyper-responsiveness.

It is anticipated that these sterically stabilized liposome carrierswill also be effective for the delivery of a wide variety of drugs forthe treatment of respiratory and other diseases. The stability of thesterically stabilized liposomes in combination with the encapsulateddrug is more pronounced than currently available drug therapies. Asdemonstrated in the following examples, this stability may allow a drug,such as a corticosteroid, to be administered only once every one to twoweeks. The dosage used in these treatments is typically the same orsimilar to that used on a daily basis. The drug may thus be administeredat two, three, four, five, six or seven days or longer intervals. Insome instances, the effective life may be up to two weeks or longer. Theeffective life of the drug in the respiratory tract has thus beenextended to the longer of at least twice the life of the drug alone, orat least one week, thus reducing the amount of the drug required toone-seventh of the previously required dosage. The term “effective life”as used herein means a period during which the drug effect is continued.Sustained action of the drug has been obtained at comparable initialdosages with a reduction in toxicity using the carrier. No suggestion orany enabling disclosure or data in the prior art is known that extendeddrug life could be obtained with these sterically stabilized liposomecarriers for aerosol drug treatments for asthma or any other disease,particularly for lung inflammation and airway hyper-responsiveness. Theextended drug life has not been obtained with the administration of thefree drug and free carrier given simultaneously but withoutencapsulation.

The drugs can be of a wide variety, such as D-4F, which is a knownanti-inflammatory cardiovascular drug for cardiovascular diseases whoseefficacy with the carrier of the present invention has been shown in theExamples for use in a mammalian respiratory tract. The drug incombination with the sterically stabilized liposomes has been shown toenter the alveoli from which oxygen is passed to the blood. It isconsidered that the drug encapsulated in the carrier is also passedthrough the tight junction from the alveoli into the blood stream as isthe oxygen. The sterically stabilized liposomes are relatively stable inthe blood stream and provide extended drug life for the encapsulateddrug.

The sterically sterilized liposome carriers of the present invention,which are adapted for combination with a variety of drugs for use in theaerosol treatment of a respiratory tract in a mammal, comprisesterically stabilized liposomes that are compatible with the respiratorytract of a mammal and which are effective to extend the effective lifeof the drug in the respiratory tract by a time equal to the longer of atleast twice the effective life of the drug alone, or at least one week.The sterically stabilized liposome carriers of the present invention aretailored to be compatible with naturally occurring fluids and surfactantfound in the lung and the liposome carriers have been observed to bindto Type 2 pneumocytes in the lungs. The carrier is tailored toaccommodate the surfactant found in the lungs so that the compositionsof the sterically stabilized liposome carriers of the present inventionare similar to lung surfactant and provide long stability to the alveoliand the respiratory tract when used to encapsulate drugs and have beenfound to be effective to extend the effective life of the drugsadministered.

The carriers have wide applicability for use in the respiratory tract ofa mammal. As shown in the examples, by electronmicroscopy observations,it has been observed that the carrier which encapsulates the drug is notdestroyed in the lungs rapidly and is deposited in the cells around thealveoli in the lung tissues (FIGS. 38 and 40). These Figures show thatthe carrier particles bearing the drug pass through the lung airwaysdown to the alveoli and the alveolar tight junctions. Since the carrierencapsulating the drug is deposited in the lung tissues, theencapsulated drug has the same medicinal effect as usual but since it isencapsulated in the carrier deposited in the lung tissues, it isreleased more slowly, thus providing a long-term effect. Any drug whichcan be encapsulated in the carrier is considered to provide the samelong-term effect in the respiratory tract or in the bloodstream. In theexamples it is shown that this effect has been achieved withcorticosteroids: BUD and TRI; monophosphoryl lipid A (MPL); peptides:D-4F (apo1 lipoprotein A-1 mimetic) and Serine Lung Protease Inhibitor(SLPI) for the treatment of lung inflammation and airwayhyper-responsiveness. All of these drugs when encapsulated and tested ina mammal as shown have given the extended effective treatment life.Further conventional liposomes, by comparison to sterically stabilizedliposomes, do not provide an extended drug life. Secondarily, the drugrapidly leaks out from the conventional liposomes.

The sterically stabilized liposome carriers of the present inventioncomprise phosphatidylcholine or phosphatidylglycerol and poly (ethyleneglycol) or both phosphatidylcholine and phosphatidylglycerol with poly(ethylene glycol). The phosphatidylcholine and phosphatidylglycerol maybe synthetically derived or they may be derived from chicken eggs orsoybeans. If derived from eggs they contain acyl groups having varyingnumbers of carbon atoms, dependent upon the variety and diet of thechicken that produces the eggs. The phosphatidylcholine is typicallypresent in a relatively significant quantity in the combination ofsterically stabilized liposomes.

A further component of the sterically stabilized liposome carriers ispoly(ethylene glycol), in the molecular range from about 500 to above5,000 daltons. The poly(ethylene glycol) may be present in combinationwith phosphatidylcholine, phosphatidylglycerol and lipids which mayinclude amino or other groups.

Any of the head groups (phosphatidylcholine and phosphatidylglycerol) orthe poly(ethylene glycol), may be attached to acyl groups containingfrom about 8 to about 18 carbon atoms. Preferably, from about 14 toabout 18 carbon atoms are present in the acyl groups. Such groupscomprise distearoyl, stearoyl oleoyl, stearoyl palmitoyl, dipalmitoyl,dioleoyl, palmitoyl oleoyl and dipalmitoleoyl.

If shorter chains are used, such as palmitoyl, dimyristoyl,didodecanoyl, didecanoyl or dioctanoyl, the poly(ethylene glycol)-lipidis likely to exchange into biological milieu. This may in some instancespermit the sterically stabilized liposome carrier to better partitiononto lung surfactant after sustained shedding or sustained exchangingits poly(ethylene glycol) moiety.

Desirably, the sterically stabilized liposome carriers may be tailoredto the particular mammalian lung system contemplated. It is considered,however, that such sterically stabilized liposome carriers will fallwithin the criteria defined above and hereinafter for the liposomes.

The sterically stabilized liposomes may contain at least one or both ofphosphatidylcholine and phosphatidylglycerol, and poly (ethylene glycol)distearoylphosphatidyldiethanolamine, lipid conjugated polyoxyethylene,lipid conjugated polysorbate, or lipids conjugated to other hydrophilicsteric coating molecules safe for in vivo use.

A particularly preferred carrier is phosphatidylcholine,phosphatidylglycerol, poly (ethylene glycol)distearoylphosphatidyldiethanolamine (PEG-DSPE).

The molecular weight of the phosphatidylcholine is desirably from about509 to about 791, preferably from about 677 to about 791 and morepreferably from about 734 to about 791. The molecular weight of thephosphatidylglycerol is desirably from about 520 to about 802,preferably from about 688 to about 802 and more preferably from about744 to about 802. The molecular weight of the poly(ethylene glycol)moiety is desirably from about 851 to about 5802, preferably from about1019 to about 3775 and more preferably from about 2749 to about 2806.The control of the molecular weight of the phosphatidylcholine and thephosphatidylglycerol is an important feature of Applicants' invention.

Some suitable carrier composition ranges are shown below in tabular form

Carrier 1 PC PG PEG-PE (mole %) (mole %) (mole %)    0-99.4    0-99.40.5-10  Preferred 60-90 10-40 1-5 Most Preferred 70-80 20-30 2-5

Carrier 2 PC PG PEG-PE CHOL (mole %) (mole %) (mole %) (mole %)   0-99.4    0-99.4 0.5-10  0.5-33 Preferred 60-90 10-40 1-5 0.5-20 MostPreferred 70-80 20-30 2-5 0.5-10

Composition 1 PC PG PEG-PE* DRUG (mole %) (mole %) (mole %) (mole %)   0-98.5    0-98.5 0.5-10  1-33 Preferred 60-90 10-40 1-5 1-33 MostPreferred 70-80 20-30 2-5 1-33 *The molecular weight of the PEG islimited to 350.

Composition 2 PC PG PEG-PE* CHOL DRUG (mole %) (mole %) (mole %) (mole%) (mole %)  0.1-99.4  0.1-99.4 0.5-10  0.1-33 1-33 Preferred 60-9010-40 1-5 0.1-20 1-33 Most Preferred 70-80 20-30 2-5 0.1-10 1-33 *Themolecular weight of the PEG is limited to 350.

Many of the commonly used sterically stabilized liposomes used forintravenous treatments are not suitable for use in the lungs. Forinstance distearoylphospatidylcholine, which has a high gel-liquidcrystalline phase transition temperature of about 54° C., is a commonlyused primary phospholipid in sterically stabilized liposomes forintravenous treatment.

The gel-liquid crystalline phase transition temperature of the mixedphospholipids in the sterically stabilized liposome carrier should be inthe range from about −20° C. to about 44° C. and preferably from about−10 to about 42° C. It is expected that for sterically stabilizedliposome carriers containing cholesterol, the transition range will bebroadened compared to that of sterically stabilized liposome carrierscontaining phospholipids alone. The inclusion of cholesterol will enablea lipid composition with a relatively high transition temperature (e.g.,in the gel phase at 37° C.) to have a substantial portion of themembrane in the fluid or liquid crystalline phase at body temperature.This is an important feature of Applicants' invention. The drugs, whichcan be encapsulated with the sterically stabilized liposome carrier ofthe present invention, comprise substantially any drug that is useful totreat diseases via the respiratory tract of a mammal. It is anticipatedthat most drugs that are useful in such treatments will be compatiblewith the sterically stabilized liposomes. Typically, the carriers andthe encapsulated drugs are administered via an aerosol to therespiratory tract.

Both the phosphatidylcholine and the phosphatidylglycerol meeting thecriteria set forth in this application can be used alone with poly(ethylene glycol) as the carrier either without or with cholesterol inamounts from about 0.1 to about 33 mole percent. The same drugs areeffective (PC and PG with PEG or PC or PG with PEG and with or withoutcholesterol) in the ranges in the Tables above as long as the criteriain this application are met to insure compatibility with the mammalrespiratory tract.

Types of drugs that can be included in the sterically stabilizedliposome carriers are not limited so long as the formation and stabilityof an encapsulation in the sterically stabilized liposome carrier is notadversely affected.

The combined sterically stabilized liposome carriers and drugs areprepared to have unilamellar or multilamellar vesicles of sizes up toabout 0.05 to about 5 microns. Preferably the composition is prepared tohave substantially homogeneous sizes in a selected size range, with theaverage diameter typically being up to about 0.02 to about 2.5micrometers. One method for obtaining the desired size is extrusion ofthe composition through polycarbonate membranes having pores of aselected size, such as up to about 0.05 to about 5 micrometers.

Some drugs that are effective for treatment of the respiratory tract ofa mammal, and are considered particularly suitable are inhaledcorticosteroids; such as, budesonide, flunisolide, triamcinolone,beclomethasone, fluticasone, mometasone, dexamethasone, hydrocortisone,methylprednisolone, prednisone, cortisone, betamethasone, or the like.Some other suitable drugs are bronchodilators; such as terbutaline,albuterol, ipatropium, pirbuterol, epinephrine, salmeterol,levalbuterol, formoterol, or the like. Chromolyn sodium and albuterolsulfate are also suitable drugs.

Other drugs that are also considered to be effective and suitablyadministered using the sterically stabilized liposome carriers of thepresent invention include, but are not limited to, leukotrieneinhibitors; such as montelukast, zafirlukast, zileuton, or the like, aswell as antihistamines; such as loratadine, cetirizine or the like.Anti-Tuberculosis drugs for MTB or atypical mycobacteria; such as,isoniazid, ethambutol, pyrazinamide, rifamycin; rifampin, streptomycin,clarithromycin, or the like, are also considered to be suitable. Otherdrugs; such as the serine lung protease inhibitor (SLPI), azelastine,and theophylline; and other peptides, such as those that relate toAllergy Immunotherapy for indoor and outdoor allergens, or the like, mayalso be considered suitable. Additionally, amikacin, gentamicin,tobramicin, rifabutin, rifapentine, sparfloxacin, ciprofloxacin,quinolones, azithromycin, erythromycin, or the like, are considered tobe suitable. Monophosphoryl lipid A (MPL) is a suitable drug.

Most previously disclosed attempts using liposomes have been withconventional liposomes and only a select few with sterically stabilizedliposomes have been used in attempts to extend the effective life ofselect drugs used in the bloodstream of mammals. These liposomes mustexist in a radically different environment than in the respiratory tractof a mammal. Particularly in the lungs, certain surfactant requirementsexist for materials which are compatible with the fluids in the lungsand the like as discussed above. Further the conventional liposomecarriers delivered to the lungs are susceptible to attack by phagocyticcells as are conventional liposomes used to position drugs in thebloodstream, which are eventually cleared mostly by liver and spleenmacrophages. Further most uses of liposomes encapsulating drugs in thebloodstream are administered via intravenous injections. While it is notclear what mechanisms exist that permit sterically stabilized liposomesto exist for longer periods of time in certain portions of the body thanwould be anticipated for liposomes that were not sterically stabilized,it is clear that the sterically stabilized liposome carriers of thepresent invention are remarkably stable in the respiratory tractenvironment and are effective to greatly extend the effective life ofencapsulated drugs used to treat various diseases of the respiratorytract.

The preparation of the sterically stabilized liposome carriers, theencapsulation and use of corticosteroid drugs, a peptide (D-4F), aprotease inhibitor (SLPI) and monophosphoryl lipid A (MPL) in thecarrier, and treatments of mice according to the present invention, aredemonstrated in the following examples to show the broad applicabilityof the carrier.

METHODS

Animals

Six week-old male C57 black 6 mice were purchased from Charles RiverLaboratories, Inc., Wilmington, Mass. The animals were provided with anovalbumin-free diet and water ad libitum and were housed in anenvironment-controlled, pathogen-free animal facility. All animalprotocols were approved by the Animal Care Committee of the Universityof Illinois at Chicago, the Medical College of Wisconsin and theZablocki Veterans Administration Medical Center, and were in agreementwith the National Institute of Health's guidelines for the care and useof laboratory animals.

Ovalbumin Sensitization

The animals were sensitized with ovalbumin (OVA). On day 0, each mousewas anesthetized with methoxyflurane given by inhalation. A fragmentedheat-coagulated OVA implant was inserted subcutaneously on the dorsalaspect of the cervical region.

For a ten-day period (days 14-24), each mouse was given a 30-minuteaerosolization of a 6% OVA solution on alternate days. This method ofsensitization led to significant elevations in eosinophil peroxidase(EPO), peripheral blood (PB) eosinophils, and serum IgE levels, alongwith lung inflammation as seen on histophathology by day 24.

Each of the nebulization doses was given at a volume of 1 milliliter for2 minutes through use of a chamber in which the mouse was allowed tobreathe freely. All treatment groups were compared with eithersensitized untreated (SENS) or unsensitized (Normal) mice.

Drugs and Reagents

BUD for daily therapy was diluted from premixed vials (0.25 mg/ml)commercially available from AstraZeneca Pharmaceutical, Wayne, Pa., andwas administered via a Salter Aire Plus Compressor, Salter Labs, Irvine,Calif. BUD for encapsulation andN-2-hydroxethylpiperzine-N′-2-ethanesulfonic acid (HEPES) was purchasedfrom Sigma Chemical, St. Louis, Mo. Phosphatidylcholine (PC),phosphatidylglycerol (PG), and poly (ethyleneglycol)-distearoylphosphatidylethanolamine (PEG-DSPE) were obtained fromAvanti Polar Lipids, Alabaster, Ala. Cholesterol was purchased fromCalbiochem, La Jolla, Calif. NaCl and KCl were purchased from FisherScientific, Pittsburgh, Pa.

Liposome Preparation

Budesonide (BUD) was encapsulated into either sterically stabilizedphosphatidylglycerol [PG]:phosphatidylcholine[PC]:cholesterol:poly(ethylene glycerol)[PEG]distearoylphosphatidylethanolamine [DSPE]-[PG:PC:Cholesterol:PEG-DSPE](2:8:5:0.5) in the sterically stabilized liposomes or conventional(phosphatidylglycerol-phosphatidylcholine-cholesterol) (2:8:5) as acarrier through use of a modified protocol derived from the protocoldescribed by Gangadharam, et al., Antimicrob Agents Chemother,1995:39:725-730.

Triamcinolone (TRI) was encapsulated into either sterically stabilizedphosphatidylglycerol [PG]:phosphatidylcholine [PC]:cholesterol:poly(ethylene glycerol) [PEG] distearoylphosphatidylethanolamine[DSPE]-[PG:PC:Cholesterol:PEG-DSPE] (2:8:5:0.5) sterically stabilizedliposomes.

A portion of the cholesterol used in control liposomes was replaced byBUD or TRI dissolved in chloroform-methanol (2:1) during the preparationof the lipid mixture. The resulting composition wasPG:PC:Cholesterol:PEG-DSPE:BUD (2:8:3:0.5:2). Lipids were dried onto thesides of a round-bottomed glass flask or glass tube by rotaryevaporation. The dried film was then hydrated by adding sterile 140mmol/L, NaCl and 10 mmol/L HEPES (pH 7.4) and vortexing.

The resulting multilamellar liposome preparations were extruded 21 timesthrough polycarbonate membranes (either 0.2 or 0.8 μm in pore diameter),(Nuclepore, Pleasanton, Calif.) through use of an Avestin extrusionapparatus, Toronto, Canada. The control carriers were prepared the sameway and of the same composition except that no BUD was added. Theresulting multilamellar liposome preparation contained about 96.8 weightpercent water and was diluted to suitable concentration (20 μg/ml) foradministration by nebulization for use.

Liposomes without cholesterol were prepared in a similar manner, exceptthat the molar ratio of the lipids was PG:PC:PEG-DSPE (2:8:0.5).

Liposomes containing MPL were prepared in a similar manner, except thatthe molar ratio of the lipids was PG:PC:MPL:PEG-DSPE (2:8:0.1:0.5).Liposomes containing both MPL and budesonide were also prepared, in theratio PG:PC:BUD:MPL:PEG-DSPE (2:8:2:0.1:0.5). Both liposomes wereextruded 21 times through polycarbonate membranes with a pore diameterof 0.8 μm.

Liposomes containing SLPI were prepared by first drying the lipids at amolar ratio of PG:PC:PEG-DSPE (2:8:0.5), and then hydrating the lipidsin sterile 140 mmol/L, NaCl and 10 mmol/L HEPES (pH 7.4) containing 1mg/ml SLPI, and proceeding with the extrusion step as above.

Liposomes containing D-4F were prepared by hydrating PG:PC:PEG-DSPE(2:8:0.5) or control PG:PC (2:8) liposomes in Hepes-buffered salinecontaining the peptide D-4F at a D-4F:lipid molar ratio of 1:40, andthen extruding the liposomes 21 times through polycarbonate membranes of0.2 μm pore diameter.

The amounts of lipid used for the Wk-Empty-S group were based on theamount of lipid nebulized for each of the BUD-encapsulated liposomes(1.39 μmol for the sterically stabilized liposomes and 3.19 μmol for theconventional liposomes).

The dose of BUD chosen was based on preliminary dose-response studieswith 5 to 50 μg of BUD as follows.

Each day, 5, 10, 15, 20 or 50 μg of BUD was administered vianebulization to groups of sensitized mice, and the dose-dependenteffects on the inflammatory parameters were evaluated. These data werecompared with data for either a group of sensitized untreated mice (SENSgroup) or a group of unsensitized mice (Normal group). A 20 μg/m/dose ofBUD was shown on histopathologic examination to effectively decrease EPOactivity in bronchoalevolar lavage fluid (BAL), PB eosinophils andinflammation of the lung tissues, along with other inflammatoryparameters, without evidence of toxicity to the spleen, liver, bonemorrow or gastrointestinal tract. In addition, there were no granulomasor abnormalities in any of the tissues evaluated.

Histopathology Observations

Histopathological examinations performed with and without Methacholinechallenge are as follows:

The lungs were removed and fixed with 10% phosphate buffered formalin.Tissue samples were taken from the trachea, bronchi, large and smallbronchioles, interstitium, alveoli, and pulmonary blood vessels. Thetissues were embedded in paraffin, sectioned at 5 μm thickness andstained with hematoxylin and eosin and analyzed using light microscopyat 100× magnification.

Coded slides were examined by a veterinary pathologist in a blindedfashion for evidence of inflammatory changes, including bronchiolarepithelial hyperplasia and wall thickening, bronchiolar, peribronchiolarand perivascular edema and accumulation of eosinophils, neutrophils, andmononuclear inflammatory cells. Each of the parameters evaluated wasgiven an individual number score. Objective measurements ofhistopathological changes include number of eosinophils surrounding thebronchi, aggregation of eosinophils around blood vessels (perivascular),accumulation of other inflammatory cells, presence of desquamation andhyperplasia of the airway epithelium, mucus formation in the lumen ofthe airways and infiltration of inflammatory cells surrounding thealveoli.

Quantitative Histopathology Scoring System LARGE SMALL ALVEOLAR TRACHEABRONCHI BRONCHIOLES BRONCHIOLES INTERSTITIUM Alveoli EpitheliumEpithelium Epithelium Epithelium Thickening(mm) Thickening(mm)Hyperplasia(mm) Hyperplasia(mm) Hyperplasia(mm) Hyperplasia(mm)Edema(mm) Edema(mm) Cells(#)-PMNs(#), Cells(#)-PMNs(#), DesquamationDesquamation Desquamation Desquamation Eosinophils(#) Eosinophils(#)Submucosa Submucosa Submucosa Submucosa Multinucleated- Edema(mm)Edema(mm) Edema(mm) Edema(mm) Microgranulomas Giant Cells(#)-PMNs(#),Cells(#)-PMNs(#), Cells(#)-PMNs(#), Cells(#)-PMNs(#), Cells(#)-PMNs(#),Cells(#) Eosinophils(#) Eosinophils(#) Eosinophils(#) Eosinophils(#)Eosinophils(#) Multinucleated-Giant Blood Vessels Granulomas GranulomasGranulomas Granulomas Cells(#) Perivascular edema Perivascular cuffingBlood Vessels Blood Vessels Blood Vessels Blood Vessels Blood VesselsCells(#)-PMNS(#), Perivascular edema Perivascular edema Perivascularedema Perivascular edema Perivascular edema Eosinophils(#) Perivascularcuffing Perivascular cuffing Perivascular cuffing Perivascular cuffingPerivascular cuffing Cells(#)-PMNS(#), Cells(#)-PMNS(#),Cells(#)-PMNS(#), Cells(#)-PMNS(#) Cells(#)-PMNS(#), Eosinophils(#)Eosinophils(#) Eosinophils(#) Eosinophils(#) Eosinophils(#)

Each of the parameters evaluated were given an individual number score.The cumulative score was obtained using the individual scores anddesignated as no inflammation (0), mild inflammation (1-2), moderateinflammation (3-4), and severe inflammation (5-6). (mm=millimeter)

Eosinophil Peroxidase (EPO) Activity in Bronchoalveolar Lavage (BAL)Fluid and Peripheral Blood (PB) Eosinophils

EPO activity was measured in the BAL. In some experimental groups EPOactivity was obtained with and without Mch challenge. At the time ofsacrifice, the trachea was exposed and cannulated with a ball-tipped24-gauge needle. The lungs were lavaged three times with 1 ml PBS. Allwashings were pooled and the samples were frozen at −70° C. The sampleswere later thawed and assayed to determine EPO activity. EPO in the BALwas assessed as follows. A substrate solution consisting of 0.1 mol/Lsodium citrate, O-phenylenediamine, and H₂O₂ (3%), pH 4.5 was mixed withBAL supernatants at a ratio of 1:1. The reaction mixture was incubatedat 37° C. and the reaction was stopped by the addition of 4 N H₂SO₄.Horseradish peroxidase was used as a standard EPO activity (ininternational units per milliliter) and was measured byspectrophotometric analysis at 490 nm.

The percentages of eosinophils were obtained by counting the number ofeosinophils in 100 white blood cells under a high-power field scope(×100) from the PB smears stained with Wright-Giemsa stain.

Total Serum IgE

Ninety-six well flat bottom plates (Fisher Scientific) were coated with100 μL per well of 2 μg/ml rat antimouse IgE monoclonal antibody (BD,PharMingen, San Diego, Calif.), and incubated overnight at 4° C. Serumwas added at a dilution of 1:50 and incubated overnight at 4° C.Purified mouse IgE (k isotype, small b allo-type anti-TNP: BDPharMingen) was used as the standard for total IgE. The samples wereincubated for one hour with biotin-conjugated rate antimouse IgE(detection antibody purchased from Southern Biotechnology, Birmingham,Ala.).

15-Lipooxyeenase (15-LO) Activity

Measurement of 15-lipooxygenase expression was performed on fresh lunghomogenates by Western blot analysis. D-4F is known to bind 15-HETE,thereby decreasing proinflammatory effects of 15-lipooxygenase activity.

Electron Microscopy Studies

Electron Microscopy studies were performed using standard protocols forpreparation and reading of the slides

Airway Hyperresponsiveness (AHR) to Methacholine (Mch)-Methods

The effectiveness of the Drug and Carrier combination on airwayreactivity or airway hyperresponsiveness (AHR) to Methacholine challenge(Mch) was evaluated by assessing Pulmonary Mechanics. These experimentsare designed to demonstrate that the sensitivity of the airway thatcauses excessive coughing or reactivity (AHR) and the like in asthmasufferers are effectively treated by the use of our Drug/Carriercombination comprising of sterically stabilized liposomes. PulmonaryMechanics were evaluated as follows:

Animals were sensitized using ovalbumin sensitization as described aboveunder the Animals section.

Comparison of C57/B16, A/J, and BALBc Mice

Using our method of ovalbumin-sensitization, C57/B16, A/J, and BALBcmice were compared in their AHR to Mch challenge, since previous studieshave demonstrated an interstrain variability in AHR to Mch challenge.There was no significant strain difference in AHR to Mch challengebetween the sensitized C57/B16 and A/J or BALBc mice. Therefore, thisstudy was conducted with C57/B16 mice.

AHR to Mch Challenge

Pulmonary resistance measurements were made after four weeks of therapy.As an antigen challenge and to demonstrate sensitization, an aerosolizeddose of 6% ovalbumin was given to each animal 24 hours before theevaluation of the pulmonary mechanics.

The animals were anesthetized with an intraperitoneal injection of asolution of ketamine and xylazine (40 mg/kg body weight for each drug).A 20 mg/kg body weight maintenance dose of pentobarbital sodium wasgiven before placement in the body plethysmogragh. The doses weretitrated to maintain a steady level of anesthesia without causingsignificant respiratory depression.

A tracheotomy was performed and a tracheotomy tube was placed in eachanimal. A saline-filled polyethylene tube with side holes was placed inthe esophagus and was connected to a pressure transducer for measurementof pleural pressure. The mice were then placed in a body plethysmographchamber for measurements of flow, volume, and pressure.

The tracheostomy tube was connected to a tube through the wall of theplethysmograph allowing the animal to breathe room air spontaneously.The esophageal catheter was connected to a pressure transducer. Properplacement of the esophageal catheter was verified using assessments ofpressure-volume-flow loops. A screen pneumotachometer and a Valadynedifferential pressure transducer were used to measure flow in and out ofthe plethysmograph.

The frequency response of the plethysmograph-pneumotachometer systemdetermined using the volume oscillator of an ElectromechanicalMultifunction Pressure Generator available from Millar Instruments,Inc., Houston, Tex., was such that the amplitude decreased by less than10% to a frequency of 12 Hz. The maximum breathing frequency in the micestudied was 4.3 Hz.

Signals from the pressure transducer and the pneumotachometer wereprocessed using a Grass polygraph (Model 7) recorder. The flow signalwas integrated using a Grass polygraph integrator (Model 7P10) tomeasure corresponding changes in pulmonary volume. Pressure, flow andvolume signal outputs were digitized and stored on computer using ananalog-to-digital data acquisition system (CODAS—available from DataqInstruments, Inc., Akron, Ohio). The pressure and volume signals werealso displayed to verify catheter placement and monitor the animalduring the experiment.

The digitalized data were analyzed for dynamic pulmonary compliance,pulmonary resistance, tidal volume, respiratory frequency and minuteventilation from about six to ten consecutive breaths at each recordingevent. Compliance and resistance were calculated from pleural pressure,airflow, and volume data.

To correct for the resistance of the tracheal cannula, the pressure-flowcurve relationship for the cannula alone was measured. It was found tohave resistance of 0.3 cmH₂)·mol⁻¹·s, which was then subtracted from thetotal resistance, measured with the animal in place to determine thepulmonary resistance. Mch challenge was performed after baselinemeasurements were obtained. Mch (Sigma Chemicals, St. Louis, Mo.) wasinjected intraperitoneally at three-minute intervals in successivecumulative doses of 30, 100, 300, 1,000 and 3,000 μg.

Study Groups

Therapy was initiated on day 25, the day after the OVA sensitization wascompleted. Sensitized animals received nebulized treatments for fourweeks. Each study group consisted of 20 mice and was followed for afour-week period. Five animals from each treatment group and from eachof the two control groups, sensitized and unsensitized, were euthanizedby means of an overdose of methoxyflurane inhaled 24 hours after thefirst treatments were given and then at weekly intervals for four weeks.At each time point, measurements of EPO in BAL, PB eosinophils, andtotal serum IgE levels were obtained and histopathologic examination ofthe lung tissues was performed.

Data Analysis

One-way ANOVA with Tukey-Kramer multiple comparison data analysis wasused for Mch responses using SigmaStat Statistical Software (SPSSScience). EPO activity analysis was performed using the Student t test.Over the Study period, there were no significant increases or decreasesin inflammation within each group according to weekly measurements forall of the inflammatory parameters being evaluated. Therefore all theweekly measurements are presented as Cumulative data and are presentedas mean+/−standard error of the mean (SEM). A p<0.05 was considered tobe statistically significant for all of the above statisticalcomparisons.

EXAMPLES

BUD 1: Comparison of BUD in the Carrier with Conventional Liposomes(FIGS. 1-5)

NORMAL Unsensitized, Untreated Normal mice SENS Sensitized, UntreatedAsthmatic mice Daily BUD 20 μg of budesonide without Carrier givendaily-Standard therapy WK-S-BUD 20 μg of budesonide in the Carrierwithout Cholesterol given once a week WK-C-BUD 20 μg of budesonideencapsulated in Conventional Carrier without Cholesterol given once aweek WK-ES Buffer loaded empty Carrier without Cholesterol or drug givenonce a week WK-BUD 20 μg of budesonide without Carrier given once a week

Histo EPO PB Eos IgE levels (Fig 2) (Fig 3) (Fig 4) (Fig 5) NORMAL — — —— SENS ↑ ↑ ↑ ↑ Daily BUD ↓ ↓ ↓ ↓ WK-S-BUD ↓ ↓ ↓ ↓ WK-C-BUD Ø Ø Ø Ø WK-ESØ Ø Ø Ø WK-BUD Ø Ø Ø Ø Legend ↑ Ø ↓ — Moderate- No significantSignificant No Severe reduction in reduction in inflammationinflammation inflammation inflammation

FIG. 1: Histopathology Picture. Representative specimens stained withhematoxylin-eosin are shown of Budesonide (BUD) treatment. a) NORMAL b)SENS c) WK-S-BUD d) Daily BUD e) WK-C-BUD f) WK-BUD. Originalmagnification×100. The lung tissues from SENS group had persistent andsignificant inflammation compared to NORMAL group. There weresignificant reductions in lung inflammation when one dose of budesonide(BUD) encapsulated in the carrier was given once a week.

Summary for BUD 1 (FIGS. 1-5):

In the set of data given for BUD 1, it was demonstrated that one dose ofbudesonide (BUD) encapsulated in the Carrier given once a week(WK-S-BUD), significantly reduced lung inflammation as shown by the lunghistology pictures (FIG. 1) and histopathology scores (FIG. 2) as wellas the lung eosinophil peroxidase activity (EPO) (FIG. 3), serum IgElevel (FIG. 4), and peripheral blood eosinophil counts (FIG. 5), aseffectively as the same dosage of BUD given once a day (Daily BUD) whencompared to the Sensitized Untreated Asthmatic group (SENS) and wascomparable to the NORMAL group. Weekly treatments with free BUD withoutCarrier (WK-BUD), BUD encapsulated in Conventional Carrier (WK-C-BUD)and Empty Carrier (WK-ES) did not have comparable effects.

BUD 2: Comparison of BUD in the Carrier with Free Drug/Free CarrierWithout Encapsulation Given Simultaneously (FIGS. 6-8).

NORMAL Unsensitized, Untreated Normal mice SENS Sensitized, UntreatedAsthmatic mice Daily BUD 20 μg of budesonide without the Carrier givendaily- Standard therapy WK-S-BUD 20 μg of budesonide in the Carrierwithout Cholesterol given once a week WK-ES Buffer loaded Empty Carrierwithout Cholesterol or drug given once a week WK-BUD 20 μg of budesonidewithout the Carrier given once a week WK-BUD & ES WK-ES and WK-BUDwithout encapsulation in the Carrier, given once a week

EPO PB Eos IgE levels (Fig 6) (Fig 7) (Fig 8) NORMAL — — — SENS ↑ ↑ ↑Daily BUD ↓ ↓ ↓ WK-S-BUD ↓ ↓ ↓ WK-ES Ø Ø Ø WK-BUD Ø Ø Ø WK-BUD & ES Ø ØØ Legend ↑ Ø ↓ — Moderate- No significant Significant No Severereduction in reduction in inflammation inflammation inflammationinflammation

Summary for BUD 2:

In the set of data given for BUD 2, it was demonstrated that one dose ofbudesonide (BUD) encapsulated in the Carrier given once a week(WK-S-BUD), reduced lung inflammation, lung eosinophil peroxidaseactivity (EPO) (FIG. 6), serum IgE levels (FIG. 8), and peripheral bloodeosinophil (FIG. 7) counts, as effectively as the same dosage of BUDgiven once a day (Daily BUD) when compared to the Sensitized UntreatedAsthmatic group (SENS) and was comparable to the NORMAL group. Weeklytreatments with only free BUD without Carrier (WK-BUD), Empty Carrier(WK-ES), or free BUD (WK-BUD) and Empty Carrier (WK-ES) givensimultaneously did not have comparable effects.

BUD 3: Comparison of BUD Encapsulated in the Carrier With and WithoutCholesterol (FIGS. 9-15).

NORMAL Unsensitized, Untreated Normal mice SENS Sensitized, UntreatedAsthmatic mice Daily BUD 20 μg of budesonide without the Carrier givendaily- Standard therapy WK-S-BUD + 20 μg of budesonide in the Carrierwith Cholesterol given (plus Chol) once a week WK-S-BUD − 20 μg ofbudesonide in the Carrier without Cholesterol (minus Chol) given once aweek WK-ES − Buffer loaded empty Carrier without Cholesterol or druggiven once a week WK-BUD 20 μg of budesonide without the Carrier givenonce a week

BUD 3: Results

FIG. 9 (A-L):

Histopathology of the airway hyperreactivity (AHR) responses without(SAR) and with (AR) Methacholine (Mch) challenge with Budesonide (BUD)treatment. Representative specimens are stained with hematoxylin-eosinare shown at a magnification of 100×. a) Normal-SAR b) Normal-AR c)SENS-SAR d) SENS-AR e) WK-S-BUD-SAR f) WK-S-BUD-AR g) Daily BUD-SAR h)Daily BUD-AR i) WK-BUD-SAR j) WK-BUD-AR k) WK-ES-SAR 1) WK-ES-AR.Original magnification×100. The lung tissues from SENS group hadpersistent and significant inflammation compared to NORMAL group withand with Mch challenge. There were significant decreases in lunginflammation only in the WK-S-BUD with and without Mch challenge. Onlywith the WK-S-BUD and the Normal groups the lung inflammation did notincrease significantly with the Mch challenge. There was a significantincrease in lung inflammation with Mch challenge with all the othergroups including the Daily BUD group.

Histo EPO PB Eos IgE levels Chol (+) FIG. 2 Chol (+) FIGS.-3, 6 Chol (+)FIGS.-4, 7 Chol (+) FIGS. 5, 8 Chol (−) FIG. 10 Chol (−) FIG. 11 Chol(−) FIG. 12 Chol (−) FIG. 13 NORMAL — — — — SENS ↑ ↑ ↑ ↑ Daily BUD ↓ ↓ ↓↓ WK-S-BUD + ↓ ↓ ↓ ↓ WK-S-BUD − ↓ ↓ ↓ ↓ WK-ES − ∅ ∅ ∅ ∅ WK-BUD ∅ ∅ ∅ ∅EPO EPO Chol (+) Chol (+) NO Mch With Mch (FIG. 14) (FIG. 14) NORMAL — —SENS ↑ ↑ Daily BUD ↓ ∅ WK-S-BUD + ↓ ↓ WK-ES − ∅ ∅ WK-BUD ∅ ∅ Histo HistoEPO EPO Chol (−) Chol (−) Chol (−) Chol (−) NO Mch With Mch NO Mch WithMch (FIG. 10) (FIG. 10) (FIG. 15) (FIG. 15) NORMAL — — — — SENS ↑ ↑ ↑ ↑Daily BUD ↓ ∅ ↓ ∅ WK-S-BUD + ↓ ↓ ↓ ↓ WK-ES − ∅ ∅ ∅ ∅ WK-BUD ∅ ∅ ∅ ∅Legend ↑ ∅ ↓ — Moderate- No significant Significant No Severe reductionin. reduction in inflammation inflammation inflammation inflammation orAHR or AHR or AHR or AHR With or No Mch challenge FIG. 14 = WithCholesterol FIGS. 10 & 15 = Without Cholesterol

Summary for BUD 3:

In the set of data given for BUD 3, it was demonstrated that BUDencapsulated in the Carrier with (WK-S-BUD+) or without Cholesterol(WK-S-BUD--) given once a week, reduced lung inflammation, lungeosinophil peroxidase activity (EPO), serum IgE levels, and peripheralblood eosinophil counts, and airway hyperreactivity (AHR) tomethacholine (MCH) challenge as effectively as the same dosage of BUDgiven once a day (Daily BUD), when compared to the Sensitized UntreatedAsthmatic group (SENS) and, was comparable to the NORMAL group. Only theWK-S-BUD+ and WK-S-BUD-- treated groups significantly reduced theEosinophil Peroxidase Activity and AHR with MCH challenge. BUD in theCarrier without Cholesterol (WK-S-Bud--) was equally effective as BUDencapsulated in the Carrier with Cholesterol (WK-S-BUD+). Weeklytreatments with only free BUD without Carrier (WK-BUD) and Empty Carrierwithout cholesterol (WK-ES--) did not have comparable effects on lunginflammation or AHR as the WK-S-BUD+ and WKS-BUD-- treated groups.

BUD 4: Comparison of BUD in the Carrier With and Without MPL (FIGS.16-18).

BUD 4: Treatment Groups

NORMAL Unsensitized, Untreated Normal mice SENS Senstitized, UntreatedAsthmatic mice Daily BUD 20 μg of budesonide without the Carrier givendaily- Standard therapy WK-S-BUD − 20 μg of budesonide in the Carrierwithout MPL or (minus MPL) Cholesterol given once a week WK-S-BUD + 20μg of budesonide in the Carrier with MPL, without (plus MPL) Cholesterolgiven once a week WK-ES − Buffer loaded empty Carrier without MPL, drug,or Cholesterol given once a week WK-ES-MPL Buffer loaded empty Carrierwithout drug or Cholesterol, with MPL given once a week

BUD 4: Results

IgE EPO EPO EPO levels (No Mch) (With Mch) (Fig 16) (Fig 17) (Fig 18)(Fig 18) NORMAL — — — — SENS ↑ ↑ ↑ ↑ Daily BUD ↓ ↓ WK-S-BUD + ↓ ↓ ↓ ↓WK-S-BUD − ↓ ↓ WK-ES − Ø Ø WK-ES-MPL Ø Ø ↓ ↓ Legend ↑ Ø ↓ — Moderate- Nosignificant Significant No Severe reduction in reduction in inflammationinflammation inflammation inflammation or AHR or AHR or AHR or AHR

Summary for BUD 4:

In the set of data given for BUD 4, it was demonstrated that BUDencapsulated in the Carrier with (WK-S-BUD+) or without MPL (WK-S-BUD--)given once a week, reduced lung inflammation, lung eosinophil peroxidaseactivity (EPO), serum IgE levels, and peripheral blood eosinophilcounts, and airway hyperreactivity (AHR) to methacholine (Mch) challengeas effectively as the same dosage of BUD given once a day (Daily BUD)when compared to the Sensitized Untreated Asthmatic group (SENS) and wascomparable to the NORMAL group. Only the WK-S-BUD+, WK-S-BUD--, and theweekly Empty Carrier with MPL (WK-ES-MPL) treatment groups significantlyreduced the Eosinophil Peroxidase Activity and AHR with Mch challenge.BUD in the Carrier with MPL (WK-S-Bud+) was equally as effective as BUDencapsulated in the Carrier without MPL (WK-S-BUD--). Weekly treatmentswith Empty Carrier without MPL (WK-ES--) did not have comparable effectson lung inflammation or AHR as the WK-S-BUD+, WK-S-BUD--, or EmptyCarrier with MPL (WK-ES-MPL) treated groups.

BUD 5: Comparison of BUD with TRI in the Carrier (FIGS. 19-23).

BUD 5: Treatment Groups

NORMAL Unsensitized, Untreated Normal mice SENS Senstitized, UntreatedAsthmatic mice Daily BUD 20 μg of budesonide without the Carrier givendaily- Standard therapy WK-S-BUD 20 μg of budesonide in the Carrier withCholesterol given once a week WK-S-TRI-20 μg 20 μg of triamcinolone inthe Carrier with Cholesterol given once a week WK-S-TRI-40 μg 40 μg oftriamcinolone in the Carrier with Cholesterol given once a week WK-ESBuffer loaded empty Carrier without drug or Cholesterol given once aweek

BUD 5: Results

FIG. 19:

Histopathology Pictures of treatment with triamcinolone (TRI).Representative specimens stained with hematoxylin-eosin are shown. a)NORMAL b) SENS c) WK-S-TRI-20 μg d) WK-S-TRI-40 μg. Originalmagnification×100. The lung tissues from SENS group had persistent andsignificant inflammation compared to NORMAL group. There weresignificant decreases in lung inflammation in both the WK-S-TRI-20 μgand WK-S-TRI-40 μg groups when compared to the SENS group.

IgE AHR Histo EPO levels With Mch (FIG. 20) (FIG. 21) (FIG. 22) (FIG.23) NORMAL — — — — SENS ↑ ↑ ↑ ↑ Daily BUD ↓ *N/A ↓ *N/A WK-S-BUD ↓ *N/A↓ *N/A WK-S-TRI-20 μg ↓ ↓ ↓ ∅ WK-S-TRI-40 μg ↓ ↓ ↓ ↓ WK-ES ∅ *N/A ∅ ∅Legend ↑ ∅ ↓ — Moderate- No significant Significant No Severe reductionin reduction in inflammation inflammation inflammation inflammation orAHR or AHR or AHR *N/A = Data not available

Summary:

In the set of data given for BUD 5, it was demonstrated that 20 μg ofTriamcinolone (TRI) encapsulated in the Carrier (WK-S-TRI-20 μg) or 40μg of TRI encapsulated in the Carrier (WK-S-TRI-40 μg) given once aweek, reduced lung inflammation and airway hyperreactivity (AHR) tomethacholine (Mch) challenge as effectively as 20 μg of Budesonide (BUD)encapsulated in the Carrier with (WK-S-BUD) or BUD given once a day(Daily BUD) when compared to the Sensitized Untreated Asthmatic group(SENS) and was comparable to the NORMAL group. WK-S-TRI-20 μg,WK-S-TRI-40 μg, WK-S-BUD, and Daily BUD all reduced the lunginflammation. Only the WK-S-TRI-40 μg and WK-S-BUD significantly reducedthe Eosinophil Peroxidase Activity and airway hyperreactivity (AHR) withMch challenge. The WK-S-TRI-20 μg reduced the AHR to Mch challenge butwas not statistically significant. Weekly treatments with Empty Carrier(WK-ES) did not have comparable effects on lung inflammation or AHR toMch challenge.

D-4F: Effect of D-4F Encapsulated in the Carrier on Lung Inflammationand AHR (FIGS. 24-32)

NORMAL Unsensitized, Untreated Normal mice SENS Senstitized, UntreatedAsthmatic mice Daily D-4F 20 μg of D-4F without the Carrier given daily,intranasally WK-S-D-4F 20 μg of D-4F in the Carrier without Cholesterolgiven intranasally once a week

D-4F: Results

FIG. 24:

Histopathology of the airway hyperreactivity (AHR) responses without andwith Methacholine (Mch) challenge with D-4F treatment. Representativespecimens are stained with hematoxylin-eosin are shown at amagnification of 100×. Top Row represents all groups without Mchchallenge a) Normal b) SENS c) Dailey D-4F d) WK-S-D-4F. Bottom Rowrepresents all groups with Mch challenge e) Normal 0 SENS g) Dailey D-4Fh) WK-S-D-4F. The lung tissues from SENS group had persistent andsignificant inflammation compared to the NORMAL group with and withoutMch challenge. There were significant decreases in lung inflammation inboth the Dailey D-4F and WK-S-D-4F groups with and without Mchchallenge.

Histo EPO PB Eos IgE levels (FIG. 25) (FIG. 26) (FIG. 27) (FIG. 28)NORMAL — — — — SENS ↑ ↑ ↑ ↑ Daily D-4F ↓ ↓ ↓ ↓ WK-S-D-4F ↓ ↓ ↓ ↓ AHRHisto Histo EPO EPO to Mch (No Mch) (With Mch) (No Mch) (With Mch) (FIG.29) (FIG. 30) (FIG. 31) (FIG. 31) (FIG. 31) NORMAL — — — — — SENS ↑ ↑ ↑↑ ↑ Daily D-4F ↓ ↓ ↓ ↓ ↓ WK-S-D-4F ↓ ↓ ↓ ↓ ↓ Legend ↑ ∅ ↓ — Moderate- Nosignificant Significant No Severe reduction in reduction in inflammationinflammation inflammation inflammation

FIG. 32:

Western Blot analysis performed on whole lung tissues and the Gelpicture depicting the 15-lipooxygenase activity after treatment withD-4F are shown in this figure. The top row represents 15-lipooxygenaseactivity and the bottom row represents beta-actin which represents acontrol for the amount of protein extracted. Lane 1 representssensitized mice treated with D-4F encapsulated in the carrier, givenonce a week. Lane 2 represents sensitized mice treated only D-4F withoutencapsulation in the carrier, given as a daily treatment. Lane 3represents sensitized untreated mice used as a control. Lane 4represents normal which are unsensitized and untreated. The intensity ofthe band for weekly treatment with D-4F encapsulated in the carrier(Lane 1) is significantly decreased and is comparable to the band thatis found with the normal untreated, unsensitized mice (Lane 4), whencompared to the untreated, sensitized mice (Lane 3). In comparison, thedaily treatment with D-4F not encapsulated in the carrier (Lane 2) didnot significantly decrease the intensity when compared to the untreated,unsensitized normal mice (Lane 4), or the untreated, sensitized mice(Lane 3).

Summary:

In the set of data given for D-4F, it was demonstrated that D-4Fencapsulated in the Carrier with (WK-S-D-4F) given intranasally once aweek, reduced lung inflammation, lung eosinophil peroxidase activity(EPO), serum IgE levels, and peripheral blood eosinophil counts, andairway hyperreactivity (AHR) to methacholine (Mch) challenge aseffectively as the same dosage of D-4F given once a day intranasally(Daily D-4F) when compared to the Sensitized Untreated Asthmatic group(SENS) and was comparable to the NORMAL group.

SLPI: Effect of SLPI Encapsulated in the Carrier on Lung Inflammationand AHR (FIGS. 33-37)

NORMAL Unsensitized, Untreated Normal mice SENS Senstitized, UntreatedAsthmatic mice Daily SLPI 20 μg of SLPI without the Carrier givendaily-Standard therapy WK-S-SLPI 20 μg of SLPI in the Carrier withCholesterol given once a week WK-ES Buffer loaded empty, Carrier withoutdrug, with Cholesterol given once a week

PB IgE Histo EPO Eos levels AHR (Fig 33) (Fig 34) (Fig 35) (Fig 36) (Fig37) NORMAL — — — — — SENS ↑ ↑ ↑ ↑ ↑ Daily SLPI ↓ ↓ ↓ ↓ Ø WK-S-SLPI ↓ ↓ ↓↓ ↓ WK-ES Ø Ø Ø Ø Ø Legend ↑ Ø ↓ — Moderate- No significant SignificantNo Severe reduction in reduction in inflammation inflammationinflammation inflammation

Summary:

In the set of data given for SLPI, it was demonstrated that SLPIencapsulated in the Carrier with (WK-S-SLPI) given once a week, reducedlung inflammation, lung eosinophil peroxidase activity (EPO), serum IgElevels, and peripheral blood eosinophil counts, and airwayhyperreactivity (AHR) to methacholine (Mch) challenge as effectively asthe same dosage of SLPI given once a day (Daily SLPI) when compared tothe Sensitized Untreated Asthmatic group (SENS) and was comparable tothe NORMAL group. Weekly treatments with Empty Carrier (WK-ES) did nothave comparable effects on lung inflammation or AHR to Mch challenge.

FIGS. 38-40:

Electron Microscopy (EM) Studies of Lung Tissues, Transmission EMstudies were obtained on the lung epithelium and airway cells. The largecell in the center is a Type II Pneumocytes which plays a critical rolein surfactant production.

FIG. 38:

Show EM of the lung epithelium of mice treated with budesonideencapsulated in conventional liposomes. The conventional liposomesencapsulated with budesonide are not detected anywhere in the lungepithelium or the airway cells. Magnification is ×5200.

FIG. 39:

Shows EM of the lung tissues and cells on mice treated with thesterically stabilized carrier encapsulated with budesonide treated oncea week. There are swirls of the sterically stabilized liposomesencapsulated with budesonide in the Type II Pneumocytes, as shown byarrows . . . . Magnification is ×5200.

FIG. 40:

Shows the same specimen as in FIG. 39 but with higher magnification. Themicrograph shows higher magnification of the airway with Type IIPneumocytes with sterically stabilized liposomes encapsulated withbudesonide. The swirls of the liposomes with drug are shown the type IIPneumocytes, as shown by arrows. Magnification is ×7200.

In the present study, it was demonstrated that one dose of BUDencapsulated in sterically stabilized liposomes, given once per week,reduced inflammation as effectively as the same dosage of BUD given onceper day. Weekly treatments with only free BUD, BUD encapsulated inconventional liposomes and empty sterically stabilized liposomes did nothave comparable effects.

While the present invention has been described by reference to certainof its preferred embodiments, it is pointed out that the embodimentsdescribed are illustrative rather than limiting in nature and that manyvariations and modifications are possible within the scope of thepresent invention. Many such variations and modifications may beconsidered obvious and desirable by those skilled in the art based upona review of the foregoing descriptions of preferred embodiments.

The invention claimed is:
 1. A method for treating a mammal with aselected drug effective for treatment of asthma and inflammation,wherein said selected drug is selected from the group consisting of:budesonide (BUD), triamcinolone (TRI), monophosphoryl lipid A (MPL),apo1 lipoprotein A-1 mimetic (D-4F), serine lung protease inhibitor(SLPI) and combinations thereof, said method consisting essentially ofa) treating the mammal with the selected drug by forming an aerosol of acomposition consisting of a sterically stabilized liposome carrierencapsulating an effective amount of the selected drug effective fortreatment of a mammal, the sterically stabilized liposome carrierconsisting essentially of phosphatidylcholine, phosphatidylglycerol,cholesterol, and poly (ethylene glycol) distearoylphosphatidyldiethanolamine (PEG-DSPE) the composition being of a particulate size upto about 0.2 to about 5.0 microns and effective to result in deposit ofthe selected drug in the airways down to the alveoli and alveolar tightjunction and having a gel-liquid transition temperature from about −20°C. to about 44° C. and providing an effective life for the selected drugin a mammal equal to at least twice the life of a single dose of theselected drug or at least one week: and, b) allowing the mammal toinhale an effective amount of the aerosol at a selected time interval.2. The method of claim 1 wherein the carrier contains up to about 98.5mole percent phosphatidylcholine, up to about 98.5 mole percentphosphatidylglycerol, from about 0.5 to about 10 mole percent PEG-DSPEand from about 1 to about 33 mole percent of the selected drug.
 3. Themethod of claim 1 wherein the selected time interval for the effectivelife of the selected drug in the respiratory tract of the mammal is atleast two days and up to two weeks.
 4. The method claim 1 wherein thecomposition contains from about 1 to about 33 mole percent cholesterol.5. The method of claim 1 wherein the composition contains from about 60to about 90 mole percent phosphatidylcholine, from about 10 to about 40mole percent phosphatidylglycerol, from about 1 to about 5 mole percentPEG-DSPE, from about 0.1 to about 20 mole percent cholesterol and fromabout 1 to about 33 mole percent of the selected drug.